This application relates to lymphokines. In particular, it relates to cytotoxic factors secreted by lymph cells and methods for making same in recombinant cells.
Immune cells such as B cells, T Cells, natural killer cells and macrophages are known to elaborate substances that exert cytotoxic activity toward tumor cells but which are innocuous to normal cells. These substances have been variously named, for example, lymphotoxin, tumor necrosis factor, NK cell cytotoxic factor, hemorrhagic necrosis factor, macrophage cytotoxin or macrophage cytotoxic factor. At the present time the identities of the proteins associated with these names are unclear. The principal difficulties have been that the biological assays employed to detect the proteins do not discriminate among them, the proteins appear to be found in nature as aggregates or hydrolytic products, and the amounts heretofore obtained have been so small that the high degree of purification needed to fully characterize the proteins has not been reached.
Typically, such cytotoxic substances are found in the sera of intact animals, or in the culture supernatants of lymph cells or cell lines after the animals or cells had been treated with a substance known to stimulate the proliferation of immune cells (an xe2x80x9cinducerxe2x80x9d). Thereafter the serum or supernatant is recovered and assayed for cytotoxic activity towards a target tumor cell line. A standard target is L-929, a murine tumor cell line. This cell line and others used in bioassays of this type are nonspecific in their lytic response because a variety of apparently discrete lymph cell products are able to effect lysis. Similar nonspecific responses are observed in in vitro tumor necrosis assays. Thus, cytolytic assays which observe for the lysis of cell lines in vitro or tumor necrosis in vivo are inadequate to distinguish among the various cytotoxic lymph products.
Cytotoxic factors tentatively have been classified on the basis of the lymph cells from which they are induced. For example, lymphotoxin is a name commonly applied to the cytotoxic secretory products of B or T lymphocytes, or cell lines derived therefrom, while tumor necrosis factor often is used to describe cytotoxic products of macrophages or their derived cell lines. This classification system, however, has not been developed to the point where there is any assurance that only a single protein is being referred to, or that proteins referred to by different names are in fact different.
Attempts have been made to purify and characterize the cytotoxic factors secreted by each cell type. To the extent that reports vary as to a property of a cytotoxic factor, or are completely inconsistent as to a given property, it could be concluded either that the characterization was erroneous or that a plurality of discrete cytotoxic factors are secreted by each cell type. For example, the cytotoxic products derived from macrophages, monocytes or monocytic cell lines, while sometimes generally referred to as tumor necrosis factor, have been reported to have properties that appear inconsistent with a theory of a single cytotoxic product. See for example the following literature: R. MacFarlan et al., 1980, xe2x80x9cAJEBAKxe2x80x9d 58(pt 5): 489-500; D. Mannel et al., 1980, xe2x80x9cInfection and Immunologyxe2x80x9d 30(2): 523-530; H. Ohnishi et al., UK patent application 2,106,117A; and J. Hammerstrom, 1982, xe2x80x9cScand J. Immunol.xe2x80x9d 15: 311-318.
On the other hand C. Zacharchuk et al., 1983, xe2x80x9cProc. Nat. Acad. Sci. USAxe2x80x9d, 80: 6341-6345 suggest that guinea pig lymphotoxin and a cytotoxic factor from guinea pig macrophages are immunochemically similar, if not identical. Similar conclusions are advanced in Ruff et al., 1981, Lymphokines Vol. 2 pp 235-272 at pp 241-242.
The attempts at characterization of immune cytotoxic factors also have focused on using as starting material the sera or peritoneal fluid of animals that have been exposed to immunogenic antigens. These sources contain the entire cornucopia of the stressed immune system, in contrast to the product or products of a single cell type or line. The following should be consulted as examples of publications of this type: S. Green et al., 1982, xe2x80x9cJ. Nat. Cancer Inst.xe2x80x9d 68(6): 997-1003 (xe2x80x9ctumor necrosis-inducing factorxe2x80x9d); M. Ruff et al., 1980, xe2x80x9cJ. Immunologyxe2x80x9d 125(4): 1671-1677 (xe2x80x9ctumor necrosis factorxe2x80x9d); H. Enomoto et al., European Patent Application 86475 (xe2x80x9cantitumor substancexe2x80x9d); H. Oettgen et al., 1980, xe2x80x9cRecent Results Cancer Res.xe2x80x9d 75: 207-212 (xe2x80x9ctumor necrosis factorxe2x80x9d); F. Kull et al., 1981, xe2x80x9cJ. Immunol.xe2x80x9d 126(4): 1279-1283 (xe2x80x9cTumor Cell Cytotoxin:); D. Mannel et al., 1980, xe2x80x9cInfection and Immunityxe2x80x9d 28(1): 204-211 (xe2x80x9ccytotoxic factorxe2x80x9d); N. Matthews et al., 1980, xe2x80x9cBr. J. Cancer: 42: 416-422 (xe2x80x9ctumor necrosis factorxe2x80x9d); S. Green et al., 1976, xe2x80x9cProc. Nat. Acad. Sci. USA:, 73(2): 381-385 (xe2x80x9cserum factorxe2x80x9d); N. Satomi et al., 1981, xe2x80x9cJpn J. Exp. Med.xe2x80x9d 51(6): 317-322; N. Matthews, 1979, xe2x80x9cBr. J. Cancerxe2x80x9d 40: 534-539 (xe2x80x9ctumor necrosis factorxe2x80x9d); K. Haranaka et al., 1981, xe2x80x9cJpn. J. Exp. Med.xe2x80x9d 51(3): 191-194 (xe2x80x9ctumor necrosis factorxe2x80x9d); and L. Old et al., European Patent Application 90892; T. Umeda et al., 1983, xe2x80x9cCellular and Molecular Biologyxe2x80x9d 29(5): 349-352; H. Enomoto et al., 1983, European Patent Application 86,475.
Further literature which should be consulted is J. Nissen-Meyer et al., 1982, xe2x80x9cInfection and Immunityxe2x80x9d 38(1): 67-73; J. Klostergaard et al., 1981, xe2x80x9cMol. Immunol.xe2x80x9d 18(12): 1049-1054; N. Sloane, U.S. Pat. No. 4,359,415; and H. Hayashi et al., U.S. Pat. No. 4,447,355; K. Hanamaka et al., 1983, European Patent Application 90,892; and G. Granger et al., 1978, xe2x80x9cCellular Immunologyxe2x80x9d 38: 388-402.
European Patent Application Publn. No. 100641 describes a cytotoxic polypeptide which was purified substantially free of impurities from a human lymphoblastoid cell culture. This polypeptide was designated lymphotoxin, although its relationship to other reported cytotoxic polypeptides under the name lymphotoxin is conjectural. It was not known whether this was the sole cytotoxic polypeptide elaborated by immune cells, as suggested by Zacharchuk et al. (Id.), or whether it was one of a potential family of cytotoxic factors.
The polypeptide of the ""641 Application has two amino termini, a larger variant ending with Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln His Pro Lys Met His Leu Ala His Ser Thr . . . and a smaller variant with the truncated amino terminus His Ser Thr Leu Lys Pro Ala Ala . . . The amino acid sequence of the lymphotoxin of the ""641 Application is disclosed in copending U.S. Ser. No. 616,503, filed May 31, 1984, wherein the term xe2x80x9clymphotoxinxe2x80x9d is defined.
According to the prior literature the interferons, which exhibit some tumor inhibitory activity, and a poorly characterized protein having an AlaAla amino terminus (U.K. Patent Application Publn. No. 2,117,385A), were candidates for non-lymphotoxin cytotoxic factors. As will be seen, the tumor necrosis factor of this invention is not an interferon, is not lymphotoxin and does not have an AlaAla amino terminus.
It is an object of this invention (a) to conclusively determine whether or not another tumor necrosis factor than lymphotoxin exists and, if so, to identify it in such a way as to clearly distinguish other such factors; (b) to produce such a factor by methods other than induced cell culture, which is expensive and yields product which is contaminated with the inducing agent, or by induction of peripheral blood lymphocytes, which is economically impractical, poorly reproducible, and produces product contaminated with homologous cellular and plasma proteins; (c) to obtain DNA encoding such tumor necrosis factor and to express the DNA in recombinant culture; (d) to synthesize such factor in recombinant culture in the mature form; (e) to modify the coding sequence or structure of such factor; (f) to formulate such factor into therapeutic dosage forms and to administer same to animals for the treatment of tumors; and (g) to produce diagnostic reagents relating to such factor.
A cytotoxic factor has been purified to homogeneity, characterized and expressed in recombinant culture. This factor is designated tumor necrosis factor (TNF) for convenience and is defined below. It is provided in substantially homogeneous form from cell culture at a specific activity of greater than about 10 million units/mg protein, and ordinarily about 100 million units/mg.
Human tumor necrosis factor synthesized in recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of a character which are physiologically acceptable for administration to patients in concert with the tumor necrosis factor. These components ordinarily will be of yeast, procaryotic or non-human higher eukaryotic origin and present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of tumor necrosis factor absolutely free of homologous proteins. Homologous proteins are those which are normally associated with the tumor necrosis factor as it is found in nature, e.g. in cells, cell exudates or body fluids. For example, a homologous protein for human tumor necrosis factor is human serum albumin. Heterologous proteins are the converse, i.e. they are not naturally associated or found in combination with the tumor necrosis factor in question.
DNA is provided that encodes tumor necrosis factor and which, when expressed in recombinant or transformed culture, yields copious quantities of tumor necrosis factor. This DNA is novel because cDNA obtained by reverse transcription of mRNA from an induced monocytic cell line contains no introns and is free of any flanking regions encoding other proteins of the organism from which the mRNA originated.
Chromosomal DNA encoding TNF is obtained by probing genomic DNA libraries with cDNA. Chromosomal DNA is free of flanking regions encoding other proteins but may contain introns.
The isolated tumor necrosis factor DNA is readily modified by substitution, deletion or insertion of nucleotides, thereby resulting in novel DNA sequences encoding tumor necrosis factor or its derivatives. These modified sequences are used to produce mutant tumor necrosis factor and to directly express mature tumor necrosis factor. The modified sequences also are useful in enhancing the efficiency of tumor necrosis factor expression in chosen host-vector systems, e.g. by modification to conform to a host cell codon preference.
These novel DNA sequences or fragments thereof are labelled and used in hybridization assays for genetic material encoding tumor necrosis factor.
In processes for the synthesis of tumor necrosis factor, DNA which encodes tumor necrosis factor is ligated into a replicable (reproducible) vector, the vector used to transform host cells, the host cells cultured and tumor necrosis factor recovered from the culture. This general process is used to construct tumor necrosis factor having the characteristics of monocyte tumor necrosis factor or to construct novel derivatives of tumor necrosis factor, depending upon vector construction and the host cell chosen for transformation. The tumor necrosis factor species which are capable of synthesis herein include mature (valyl amino-terminal) tumor necrosis factor, pretumor necrosis factor (xe2x80x9cpreTNFxe2x80x9d, defined herein), and derivatives of TNF including (a) fusion proteins wherein TNF or any fragment thereof (including mature tumor necrosis factor) is linked to other proteins or polypeptides by a peptide bond at the amino and/or carboxyl terminal amino acids of TNF or its fragments, (b) TNF fragments, including mature tumor necrosis factor or fragments of preTNF in which any preprotein amino acid is the amino-terminal amino acid of the fragment, (c) mutants of TNF or its fragments (including mature tumor necrosis factor) wherein one or more amino acid residues are substituted, inserted or deleted, and/or (d) methionyl or modified methionyl (such as formyl methionyl or other blocked methionyl species) amino-terminal addition derivatives of the foregoing proteins, fragments or mutants.
Ordinarily, if a mammalian cell is transformed with (a) a vector containing the entire tumor necrosis factor structural gene (including a 5xe2x80x2 start codon), or (b) the gene for mature tumor necrosis factor or a tumor necrosis factor derivative operably ligated to a eukaryotic secretory leader (which may also include the tumor necrosis factor secretory leader presequence), and the cell cultured, then mature tumor necrosis factor is recovered from the culture.
Similarly, if DNA which encodes TNF is operably ligated in a vector to a secretory leader which is properly processed by the host cell to be transformed (usually the organism from which the leader sequence was obtained), the host transformed with the vector and cultured, then the tumor necrosis factor is synthesized without amino-terminal methionyl or blocked methionyl. In particular, E. coli transformed with vectors in which DNA encoding mature tumor necrosis factor is ligated 5xe2x80x2 to DNA encoding the STII enterotoxin signal polypeptide will properly process a high percentage of the hybrid preprotein to mature tumor necrosis factor. Secretory leaders and host cells may be selected that also result in proper transport of mature protein into cell periplasm.
Also within the scope of this invention are derivatives of tumor necrosis factor other than variations in amino acid sequence or glycosylation. Such derivatives are characterized by covalent or aggregative association with chemical moieties. The derivatives generally fall into three classes: salts, side chain and terminal residue covalent modifications, and adsorption complexes.
If DNA encoding mature tumor necrosis factor is operably ligated into a vector, the vector used to transform a host cell and the cell cultured, mature tumor necrosis factor is found in the cell cytoplasm. Accordingly, it is unnecessary to devise secretion systems in order to obtain mature tumor necrosis factor. This was surprising because, ordinarily, direct expression yields methionylated protein. Further, the protein is stable and soluble in recombinant cell culture, i.e., it is neither proteolytically cleaved by intracellular proteases nor deposited as refractile bodies. Accordingly, novel fermentations are provided that comprise lower eukaryotic or prokaryotic cells having unmethionylated mature tumor necrosis factor located within the cytoplasm of such cells.
While tumor necrosis factor may be prepared by culturing animal cell lines, e.g. a monocytic cell line induced by growth in the presence of 4-beta-phorbol-12-myristate-13-acetate (PMA) or immortal cell lines such as hybridomas or EBV transformed cells (U.S. Pat. No. 4,464,465), it is preferable to synthesize tumor necrosis factor in recombinant cell culture as described further below.
Once tumor necrosis factor is prepared by fermentation it generally is purified by recovering the supernatant culture fluid or lysed cell culture, removing solids, adsorbing tumor necrosis factor from the supernatant admixture, (containing tumor necrosis factor and other proteins) onto a hydrophobic substance, eluting tumor necrosis factor from the substance, adsorbing tumor necrosis factor onto a tertiary amino anion exchange resin, eluting tumor necrosis factor from the resin, adsorbing tumor necrosis factor onto an anion exchange resin (preferably quaternary amino-substituted) having substantially uniform particle size, and eluting tumor necrosis factor from the resin. Optionally, the tumor necrosis factor compositions are concentrated and purified by chromatofocusing at any point in the purification procedure, for example by isoelectric focusing or passage through a sieving gel such as Sephadex G-25.
The purified tumor necrosis factor from recombinant or induced cell culture is combined for therapeutic use with physiologically innocuous stabilizers and excipients and prepared in dosage form as by lyophilization in dosage vials or storage in stabilized aqueous preparations. Alternatively, tumor necrosis factor is incorporated into a polymer matrix for implantation into tumors or surgical sites from which tumors have been excised, thereby effecting a timed-release of the tumor necrosis factor in a localized high gradient concentration.
The compositions herein are obtained free of contaminant cytotoxic factors such as lymphotoxin, interferons or other cytotoxic proteins referred to in the literature. However, in therapeutic applications tumor necrosis factor is advantageously combined with predetermined amounts of lymphotoxin and/or interferon. Compositions containing tumor necrosis factor and an interferon such as gamma interferon are particularly useful since they have been found to exert a synergistic cytotoxic activity.
Tumor necrosis factor compositions are administered to animals, particularly patients bearing malignant tumors, in therapeutically effective doses. Suitable dosages will be apparent to the artisan in the therapeutic context, as is further described infra.